Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts
Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts
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通过激活人牙周膜成纤维细胞中的 TLR2、TLR4、NOD1 和 NOD2 表达 TRAF6 和促炎细胞因子
DOI：
10.1016/j.archoralbio.2011.02.020
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发表时间：
2011-10-01
影响因子：
3
通讯作者：
Huang, Ding-ming
中科院分区：
文献类型：
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作者：
Tang, Lu;Zhou, Xue-dong;Huang, Ding-ming
目的：人牙周膜成纤维细胞（HPDLFs）在根尖周组织中起着重要的抗菌作用。Toll样受体（TLR）和核苷酸结合寡聚化结构域（NOD）是识别微生物病原体并启动促炎信号传导的两种主要形式的先天免疫传感器。肿瘤坏死因子受体相关因子6（TRAF 6）是TLR介导的核因子-κ B（NF-κ B）信号通路激活的衔接蛋白，其诱导促炎细胞因子的产生。本研究的目的是研究TLR 2、TLR 4、NOD 1和NOD 2在HPDLFs中的表达。方法：采用逆转录-聚合酶链反应（RT-PCR）、流式细胞术和免疫组化方法检测TLR 2、TLR 4、NOD 1和NOD 2的表达。用TLR和NOD激动剂刺激HPDLF。用实时荧光定量PCR和Western blot检测TRAF 6的表达。通过酶联免疫吸附测定（ELISA）测定培养上清液中IL-1 β、IL-6和IL-8的浓度。最后，通过使用针对TRAF 6的小干扰RNA（siRNA），我们分析了HPDLFs中IL-1 β、IL-6和IL-8在TLR和NODs激动剂刺激后的产生。TRAF 6和促炎细胞因子（IL-1 β、IL-6和IL-8）的表达水平在TLR和NOD活化后显著上调。TLRs和NODs的共激活对TRAF 6和促炎细胞因子的产生具有协同作用。我们还发现TRAF 6抑制导致减少IL-1 β，IL-6和IL-8的表达TLR和NOD激动剂challenge.Conclusion：这些研究结果表明，TLR 2，TLR 4，NOD 1和NOD 2是功能性受体在HPDLFs在先天免疫反应入侵细菌，并通过TLR和NODs的信号组合导致协同增强炎症反应在HPDLFs。此外，TLR和NOD信号转导涉及TRAF 6有助于HPDLF的炎症反应。(C)2011爱思唯尔有限公司保留所有权利。
Objective: Human periodontal ligament fibroblasts (HPDLFs) play a crucial role in protecting against oral bacteria in periapical tissue. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) are two major forms of innate immune sensors that recognize microbial pathogens and initiate pro-inflammatory signalling. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein for TLR-mediated nuclear factor-kappa B (NF-kappa B) signalling pathway activation that induces the production of pro-inflammatory cytokines. The aim of this study was to investigate the expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. We also investigated the expression of TRAF6 and pro-inflammatory cytokines induced by the activation of TLRs and NODs.Methods: The expression of TLR2, TLR4, NOD1, and NOD2 was measured by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and immunostaining. HPDLFs were stimulated with TLR and NOD agonists. Then, the expression of TRAF6 was measured by real-time PCR and western blot. Concentrations of IL-1 beta, IL-6, and IL-8 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Finally, by using small interfering RNA (siRNA) for TRAF6, we analysed the production of IL-1 beta, IL-6, and IL-8 in HPDLFs upon stimulation with TLRs and NODs agonists.Results: We found clear mRNA and protein expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. The expression levels of TRAF6 and pro-inflammatory cytokines (IL-1 beta, IL-6, and IL-8) were markedly up-regulated upon the activation of TLRs and NODs. Furthermore, the co-activation of TLRs and NODs had synergistic effect on the production of TRAF6 and pro-inflammatory cytokines. We also found TRAF6 suppression resulted in reduced IL-1 beta, IL-6, and IL-8 expression upon TLR and NOD agonists challenge.Conclusion: These findings indicated that TLR2, TLR4, NOD1, and NOD2 are functional receptors in HPDLFs during innate immune responses to invading bacteria, and a combination of signalling through TLRs and NODs leads to the synergistic enhancement of inflammatory reactions in HPDLFs. In addition, TLR and NOD signalling involving TRAF6 contribute to inflammatory responses in HPDLFs. (C) 2011 Elsevier Ltd. All rights reserved.